Effect of distension on ADH-induced osmotic water flow in toad urinary bladder

Summary We recently described a method by which the resistance to water flow of the luminal membrane of ADH-stimulated toad bladder can be quantitatively distinguished from that of barriers lying in series with it. This method requires estimates of both total bladder water permeability (assessed by transbladder osmotic water flow at constant gradient) and luminal membrane water permeability (assessed by quantitation of the frequency of ADH-induced luminal membrane particle aggregates). In the present study we examined the effect of bladder distension on transepithelial osmotic water flow before and during maximal ADH stimulation. Base-line water flow was unaffected by bladder distension, but hormonally stimulated flow increased systematically as bladders became more distended. Distension had no effect on the frequency of ADH-induced intramembranous particle aggregates. By comparing the relationships between aggregate frequency and hormonally induced water permeability in distended and undistended bladders, we found that distension appeared to enhance ADH-stimulated water flow by decreasing the resistance of the series permeability barrier while the apparent water permeability associated with each single luminal membrane aggregate was unaffected. In that bladder distension causes tissue thinning, the series resistance limiting ADH-stimulated water flow appears to be accounted for by deformable barriers within the bladder tissue itself, probably unstirred layers of water.     

Abnormally spliced messenger RNA in erythroid cells from patients with β+ thalassemia and monkey cells expressing a cloned β+-thalassemic gene

The reduced β-globin synthesis characterizing the β⁺ thalassemia phenotype has been shown to be caused by anomalous processing within the small Intervening sequence (IVS1) of the β-globin mRNA precursor. The β-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3′ junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3′-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with β⁺ thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned β⁺-thalassemic gene preferentially generated the anomalously spliced RNA when expressed In monkey kidney cells. The anomalously spliced RNA constituted approximately 80%–90%, and normal β RNA approximately 10%–20%, of the total β mRNA. In contrast, the small amount of β mRNA present in reticulocytes from such patients consisted predominantly of normal β mRNA. These results suggest that the reduced amount of normally functioning β mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent Instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.     

Metronidazole and the isolation of temperature-sensitive photosynthetic mutants in cyanobacteria

A procedure has been developed for use of metronidazole (2-methyl-5-nitroimidazole-1-ethanol) as an enrichment agent during the isolation of temperature-sensitive, photosynthetic mutants in the cyanobacteriumSynechococcus cedrorum. The protocol includes incubation with this drug following mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Incubation of photosynthetically activeS. cedrorum cells with 1 mM metronidazole causes a light-dependent reduction of cell viability. Maximum reduction in cell viability occurred following 6 h of incubation. Cessation of electron transport reduced the impact of the drug by five orders of magnitude. Yet during the time of incubation, metronidazole did not influence the electron transport capacities of theS. cedrorum cells, suggesting that the thylakoid membrane was not the target of the toxic effects of this drug. In addition, this drug was found to be an effective electron acceptor to photosystem I although high concentrations were required to observe maximum rates of electron transfer. Metronidazole interacted in a noncompetitive manner with methyl viologen, which suggested that those two acceptors to photosystem I have unique reduction sites on theS. cedrorum thylakoid membrane. The temperature-sensitive strains that were isolated using the procedure presented here were assessed for photosynthetic electron transport and chlorophyll fluorescence (induction kinetics and low-temperature emission spectra) characteristics. Approximately one-half of the temperature-sensitive mutants isolated possessed abnormal photosynthetic properties when shifted to the restrictive temperature (40°C). A total of 31 strains have been characterized and initially classified, showing abnormalities throughout the photosynthetic electron-transport chain.     

Plasmodium lophurae: Antibody-induced movement and capping of surface membranes of erythrocyte-free malarial parasites

Surface antigens of the avian malarial parasite, Plasmodium lophurae, and its host cell, the duckling erythrocyte, were visualized at the ultrastructural level using rabbit antisera and ferritin-labeled goat anti-rabbit IgG. Rabbit antisera to P. lophurae caused an aggregation of parasite and parasitophorous vacuole surface membrane antigens, a phenomenon known as capping. Capping required living plasmodia and did not occur if parasites had been fixed with glutaraldehyde prior to exposure to antisera. Antisera against duckling erythrocytes did not cross-react with erythrocyte-free malarial parasites, and did not form caps on the surface of the red blood cell. Antiplasmodial sera did not react with normal or malaria-infected red cells. These results suggest that surface membrane proteins of the intracellular plasmodium are capable of lateral movement.     

Genes Affecting the Expression of Cytochrome c in Yeast: Genetic Mapping and Genetic Interactions

The four mutant genes, cyc2, cyc3, cyc8 and cyc9, that affect the levels of the two iso-cytochromes c in the yeast Saccharomyces cerevisiae have been characterized and mapped. Both cyc2 and cyc3 lower the amount of iso-1-cytochrome c and iso-2-cytochrome c; whereas, cyc8 and cyc9 increase the amount of iso-2-cytochrome c. The cyc2, cyc3, cyc8 and cyc9 genes are located, respectively, on chromosomes XV, I, II and III, and are, therefore, unlinked to each other and unlinked to CYC1, the structural gene of iso-1-cytochrome c and to CYC7, the structural gene of iso-2-cytochrome c. While some cyc3 mutants are completely or almost completely deficient in cyotchromes c, none of the cyc2 mutants contained less than 10% of parental level of cytochrome c even though over one-half of the mutants contain UAA or UAG nonsense mutations. Thus, it appears as if a complete block of the cyc2 gene product still allows the formation of a residual fraction of cytochrome c. The cyc2 and cyc3 mutant genes cause deficiencies even in the presence of CYC7, cyc8 and cyc9, which normally cause overproduction of iso-2-cytochrome c. We suggest that cyc2 and cyc3 may be involved with the regulation or maturation of the iso-cytochromes c. In addition to having high levels of iso-2-cytochromes c, the cyc8 and cyc9 mutants are associated with flocculent cells and other abnormal phenotypes. The cyc9 mutant was shown to be allelic with the tup1 mutant and to share its properties, which include the ability to utilize exogenous dTMP, a characteristic flocculent morphology, the lack of sporulation of homozygous diploids and low frequency of mating and abnormally shaped cells of alpha strains. The diverse abnormalities suggest that cyc8 and cyc9 are not simple regulatory mutants controlling iso-2-cytochrome c.     

Yeast UAA suppressors effective in psi+ strains: leucine-inserting suppressors.

We have previously reported the isolation and characterization of UAA suppressors from a haploid strain of yeast Saccharomyces cerevisiae containing the ψ⁺ non-Mendelian determinant which increases the efficiency of action of certain suppressors (Ono et al., 1979). Most of the suppressors caused the insertion of either tyrosine or serine. In contrast, the pattern of suppression of nutritional markers suggested that the rare suppressor, SUP26, inserted in an amino acid other than tyrosine or serine. In this investigation we report the characterization of additional suppressors, similar to SUP26, that were isolated on a medium lacking uracil and containing canavanine; this medium is expected to exclude serine-inserting suppressors because they do not suppress the ura4-1 marker, and to exclude tyrosine-inserting suppressors because they suppress the can1-100 marker. The total of 155 revertants similar to the SUP26 suppressor were analyzed genetically and these could be assigned to one or another of the six distinct loci SUP26, SUP27, SUP28, SUP29, SUP32 and SUP33. The SUP26, SUP27 and SUP29 loci mapped on chromosomes XII, IV and X, respectively. The detailed map position of the SUP29 suppressor suggests that it may be allelic to the SUP30 suppressor reported by Hawthorne &; Mortimer (1968). These six suppressors had the same pattern of suppression of UAA nutritional markers and all of them had a similar low efficiency of action on the iso-1-cytochrome c mutation cyc1-72. The efficiency of each of these suppressors was increased by a chromosomal allo-suppressor, sal. Each of the six suppressors caused the insertion of leucine in iso-1-cytochrome c at the UAA site of the cyc1-72 mutation. It is suggested that the gene products of these suppressors are redundant forms of the same leucine transfer RNA.     

A modified walking rhythm employed during righting behavior in the cockroachGromphadorhina portentosa

Summary Electromyograms were recorded from leg muscles of the cockroachGromphadorhina during walking and righting under free-ranging and tethered conditions. Two muscles which are essentially synergistic during walking become antagonistic during righting (Fig. 3, 4). This explains in part the difference in the direction of the leg stroke in the two behaviors (Fig. 2). Other properties of the muscle activity are very similar during the two rhythms: the same motoneurons appear to be active (Fig. 5, 6); cycle frequencies are the same; the burst length of one motoneuron studied varies with burst frequency in a generally similar manner in both behaviors (Fig. 7); inter-leg coordination is the same (Fig. 9); and transganglionic coupling characteristic of walking can occur while a leg on one side is engaged in walking, and its contralateral homologue is engaged in righting (Fig. 10). Although other properties of the leg rhythms are different in walking and righting, these differences appear to result from dissimilarities in sensory feedback. It is concluded that although the two leg rhythms are superficially quite different, the underlying central neuronal rhythms are very similar, and possibly result from activity in the same central oscillatory cell or circuit.We thank Carol Smith for technical assistance. This work was supported by NIH grant #NS09083-05. Computation was done at the New York State Veterinary College Computer Facility which is supported by NIH grant RR 326.